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Biomedical subjects

N J Schmidt

Publications and source records attributed to N J Schmidt.

At least 37 records · Page 2Linked to original sources

Rapid viral diagnosis.

There has been much interest and activity in the development of techniques for rapid viral diagnosis which would allow successful intervention in the treatment of patients or their contacts or in the control of viral diseases in the community. The greatest emphasis has been on techniques that permit viral detection directly in the clinical specimen, since these avoid the need to cultivate the agent, are feasible for detection of viruses that cannot be cultivated, and can detect virus in specimens in which the agent is no longer infectious. Direct methods used for viral detection include electron microscopy and various immunoassays which are based on demonstrating reactivity of viral antigen in the specimen with known viral antisera. The use of immunoassays for more rapid identification of viruses isolated in laboratory host systems and for selective detection of viral antigen in inoculated cell cultures even before the agents produce an observable effect has been an important advance in viral diagnosis by the approach of isolation and identification. The reliability of all specific viral identification procedures depends on the use of high quality viral antisera. Some of the problems previously encountered in preparing satisfactory viral immune reagents are being overcome through the availability of highly specific monoclonal antibodies produced by cell hybridization techniques. Virus-specific IgM antibody assays for rapid diagnosis have been improved greatly through the use of a "capture" technique in which antibody to the human mu chain is used in the solid phase to separate IgM from other serum components which might compete with IgM antibody or give nonspecific reactivity, and also through the availability of highly specific monoclonal antibodies to the human mu chain. A variety of simple assays for determination of viral antibody status have been developed, and many are commercially available. The reliability of some of these antibody assays has been improved through the incorporation of more suitable controls and through better definition of interpretations which should be made from test results.

Antibodies, Monoclonal↗

Effect of the pesticide carbaryl on replication of human and simian varicella viruses.

Pretreatment of host cells with carbaryl delayed the early spread of simian and human varicella virus infections. Toward the end of the growth cycle there was an apparent enhancement of infection, since treated cultures showed more infectious centers than did untreated ones in which infectivity had reached maximum levels and then declined.

Animals↗

Broadly reactive immunofluorescence test for measurement of immunoglobulin M and G antibodies to Ureaplasma urealyticum in infant and adult sera.

The indirect immunofluorescence test was used to measure immunoglobulin M (IgM) and IgG antibodies to acetone-fixed Ureaplasma urealyticum organisms in sera from 128 adults with genital infections and from 713 symptomatic newborns and babies 1 day to 18 months old. Thirty-four percent of the adults had demonstrable IgG antibody to ureaplasma. IgM antibody was detected in 2 of the adult sera and in 17 of the infant sera. These babies were divided into two distinct groups. Ten of the infants presented at birth with various physical findings, whereas the onset of symptoms for the other 7 occurred 3 to 13 weeks after birth, and the major clinical finding in 6 of the 7 was respiratory distress. The results of this study suggested that U. urealyticum infection may be associated with fetal damage and infant pneumonia, and if this is substantiated, the indirect immunofluorescence test employing acetone-fixed antigen to measure IgM antibody to U. urealyticum may be an important diagnostic tool.

Adult↗

Use of monoclonal antibodies to human immunoglobulin M in "capture" assays for measles and rubella immunoglobulin M.

Monoclonal antibodies to human immunoglobulin M (IgM) were used in a four-phase enzyme immunofluorescence "capture" assay for determination of IgM antibodies to measles and rubella viruses. Little or no background reactivity was seen in the test system, and interfering effects of rheumatoid factor were avoided by preabsorption of test sera with aggregated human IgG. Virus-specific IgM antibody was demonstrable in 23 of 24 patients with serological evidence of measles virus infections and in 36 of 36 patients with serological evidence of postnatal rubella infection. A few of the rubella patients did not show IgM antibody until 5 days after onset of illness. The enzyme immunofluorescence assay was able to demonstrate rubella IgM antibody in congenitally infected newborns, whereas indirect immunofluorescence results for virus-specific IgM were negative. Viral IgM antibody was not detected in persons with past infections with the test viruses, in young children without evidence of past infection, or in patients infected with heterotypic viruses, rickettsiae, chlamydiae, or mycoplasmas.

Antibodies, Monoclonal↗

Serological investigation of an outbreak of simian varicella in Erythrocebus patas monkeys.

An epizootic of simian varicella occurring in a colony of Erythrocebus patas monkeys was studied serologically by using radioimmunoassay and neutralization tests against (i) a virus strain isolated from an animal that died during the epizootic, (ii) a simian varicella virus strain from an earlier outbreak of simian varicella-like disease at another facility, and (iii) human varicella-zoster virus. Serological tests detected more cases of infection among the animals exposed to virus during the epizootic than were evidenced by clinical findings; only 6 of the 26 animals with seroconversion developed a rash. Good correlation was seen between antibody responses demonstrated by radioimmunoassay and by the neutralization tests. Specificity of the radioimmunoassay was evidenced by the complete agreement with neutralization results for 17 animals which failed to show an antibody response over the course of the outbreak and were assumed not to have been infected. Thus radioimmunoassay is a reliable, rapid, and relatively economical method which could be used for serological screening of primates entering experimental colonies to identify those which might be potential sources of outbreaks through activation of latent simian varicella virus infection. Close correlation was seen between antibody responses to the virus strain from the current outbreak and the one from another epizootic, indicating that the two outbreaks were caused by antigenically similar viruses. Animals showing neutralizing antibody responses to the simian varicella viruses also showed responses to human varicella-zoster virus, which further substantiates the close antigenic relationship between human and simian varicella viruses.

Animals↗

Further evidence for common antigens in herpes simplex and varicella-zoster viruses.

To elucidate the mechanism of heterologous antibody responses to herpes simplex virus (HSV) and varicella-zoster virus (VZV) which occur in some patients with HSV or VZV infections, stronger evidence was sought for the existence of cross-reacting antibodies to these viruses, using antibody absorption procedures. Absorption of sera from initial HSV infections with HSV antigen was found to abolish heterologous antibody titer rises to VZV, as demonstrated in complement fixation, neutralization, and anti-complement immunofluorescence test systems. In most instances, convalescent-phase titers to heterologous VZV were reduced by HSV absorption to levels comparable to those in the acute-phase serum, indicating that cross-reacting antibodies were, in fact, responsible for the heterologous antibody titer rises. Absorption of convalescent-phase sera from HSV or VZV patients with homologous antigen also abolished or greatly diminished immunoprecipitating activity with the heterologous antigen, furnishing additional evidence of the existence of cross-reacting antibodies. Absorption of sera with insolubilized IgG to re-remove rheumatoid factor, which was present in a number of the sera studied, had no effect on either homologous or heterologous antibody titer increases. The demonstration of cross-reacting antibodies to HSV and VZV supports the concept that these two human herpesviruses share common antigen(s).

Antibodies, Viral↗

Serological reactivity of some monoclonal antibodies to varicella-zoster virus.

Monoclonal antibodies to varicella-zoster virus, free from host cell reactivity, were produced by cell fusion technics. Antibodies from four different clones showed diverse activities in neutralization immunofluorescence and complement fixation assays. The antibodies provide useful reagents for viral diagnosis and viral antigenic characterization.

Animals↗

Improved yields and assay of simian varicella virus, and a comparison of certain biological properties of simian and human varicella viruses.

Studies were performed to define conditions under which propagation, assay and stabilization of the Delta herpesvirus (DHV) strain of simian varicella virus might be improved, and to compare biological properties of DHV with those of human varicella zoster virus (VZV). A mycoplasma contaminant was successfully eliminated from the DHV seed virus by treatment with a specific anti-serum. DHV was found to replicate more efficiently in the BS-C-1 line of African green monkey kidney cells than in Vero cells, and seed virus preparations in the form of virus-infected cells were produced which had infectivity titers greater than or equal to 1 X 10(6) p.f.u./ml. Greater yields of virus were produced in cultures infected as dispersed cells than as preformed monolayers. Infectious DHV could be released from host cells by sonic treatment of heavily infected cultures at 48 h post infection. Certain agents reported to enhance replication of herpes viruses (caffeine, carbaryl, the tumor promoter 12-0-tetra-decanoyl-phorbol-13-acetate, and DEAE-dextran) had no enhancing effect on replication of DHV. However, DEAE-dextran in the maintenance medium enhanced spontaneous release of DHV into culture fluids. Plaquing efficiency and plaque size of DHV were greater in BS-C-1 than in Vero cells, and plaque assays and plaque reduction neutralization tests were developed in this cell system using a solid overlay medium with neutral red vital stain. Neutralization of DHV was markedly enhanced by fresh guinea pig complement. The newly developed neutralization test demonstrated more vigorous antibody responses to DHV in active and latent VZV infections than were demonstrated with previous procedures. In addition to their preferential growth in monkey and human cells respectively, DHV and VZV were found to differ markedly in their rates of attachment to host cells, with DHV requiring over 6 h of adsorption, while VZV adsorption was essentially complete at 1 h. Also, cell-free DHV was much more resistant than cell-free VZV to repeated cycles of freezing and thawing.

Animals↗

Production of monoclonal antibodies to human IgM for assay of viral IgM antibodies.

Monoclonal antibodies to human IgM were produced by fusing the Sp 2/0-Ag 14 line of mouse myeloma cells with spleen cells from BALB/c mice immunized with purified human IgM. From 6 clones which secreted antibody to human IgM, the one which produced the highest levels of antibody and grew relatively rapidly was selected for expansion and production of immune reagents for viral IgM antibody assays. Mouse ascitic fluids produced with this clone of cells had antibody titers for human IgM of 1 X 10(-10) by indirect enzyme immunofluorescence assay (EIFA). The monoclonal antibodies were found to belong to subclass 1 of murine IgG, and their specificity was shown to be directed against the Fab portion of the mu chain of human IgM. Antibodies from murine ascitic fluid conjugated with horseradish peroxidase were shown to be suitable for assay of measles IgM antibody by indirect immunoperoxidase staining. Antibodies conjugated with alkaline phosphatase could be used in an indirect EIFA for determination of measles IgM antibodies; use of monoclonal conjugates in this system eliminated the nonspecific activity observed in tests utilizing polyclonal anti-mu reagents. Further, the monoclonal antibodies were highly satisfactory for use in a 'capture' system for viral IgM antibody assays. The availability of monoclonal antibodies to human IgM overcomes problems with specificity, consistency and supply which have previously hindered development and standardization of viral IgM antibody assays.

Animals↗

Alpha interferon administration to infants with congenital rubella.

Three infants with congenital rubella syndrome were given human leukocyte (alpha) interferon at doses of 2 x 10(5) to 7 X 10(5) U/kg per day for 10 days. A transient decrease in pharyngeal virus excretion was observed with treatment. No significant side effects were associated with the administration of human leukocyte interferon to these infants.

Humans↗

Monoclonal antibodies for rapid, strain-specific identification of influenza virus isolates.

Monoclonal antibodies conjugated with fluorescein permitted rapid, strain-specific identification of influenza A isolates and type-specific identification of influenza B isolates by direct immunofluorescence staining. Identification of H1 influenza A strains could be accomplished by direct immunofluorescence on cell culture fluids lacking sufficient hemagglutinin activity to permit identification by hemagglutination inhibition.

Animals↗

Application of immunoperoxidase staining to more rapid detection and identification of rubella virus isolates.

Efforts were made to shorten the time required for detection of rubella virus in clinical materials through the use of immunoperoxidase (IP) staining. Comparative studies were performed in which specimens were inoculated in parallel into BHK-21 hamster kidney cells, which were examined by IP staining at 5 days, and into BK-13 and BS-C-1 cells, which were examined in two ways, viz., by subpassage at 7 days into BHK-21 cells and IP staining 3 days later and by subpassage at 7 days into BS-C-1 cells followed by interference testing and immunofluorescence (IF) staining on positive materials (standard method). Direct inoculation into BHK-21 cells with IP staining at 5 days permitted detection and identification of 59% of the 63 positive specimens. Toxicity of some specimens preserved with sorbitol and of certain tissue specimens reduced the number of satisfactory examinations which could be performed in this system. Virus detection and identification by IP staining on subpassaged RK-13 and BS-C-1 materials, requiring a total of 18 days, was comparable to the longer interference-IF method, requiring 17 days. Results obtained by IP staining and interference-IF showed 98% correlation on RK-13 materials and 97% correlation on BS-C-1 materials. IP staining on inoculated BHK-21 cells can be a useful method for rapid identification of a relatively high proportion of rubella-positive specimens, particularly if sorbitol-preserved specimens are avoided, and IP staining on subpassaged RK-13 and BS-C-1 materials is a highly satisfactory alternative to the longer interference-IF method.

Animals↗

Comparison of anticomplement immunofluorescence and fluorescent antibody-to-membrane antigen tests for determination of immunity status to varicella-zoster virus and for serodifferentiation of varicella-zoster and herpes simplex virus infections.

The anticomplement immunofluorescence (ACIF) test was compared with the fluorescent antibody-to-membrane antigen (FAMA) test for determining varicella-zoster virus antibody levels as a measure of varicella-zoster virus immunity status. The ACIF test was found to be comparable to the FAMA test in sensitivity and could be used for examining sera at low dilutions of 1:2 and 1:4. In addition, the ACIF method proved to be a more economical procedure in terms of antigen required and personnel time necessary to perform the test. Heterologous varicella-zoster virus antibody titer rises were demonstrated by the FAMA test with 10 serum pairs from patients with clinically diagnosed genital herpes simplex virus infection, indicating that the FAMA test is no more suitable than other serological methods for serodifferentiation of those herpes simplex virus and varicella-zoster virus infections in which antibody increases occur to both antigens.

Antibodies, Viral↗

Humoral immune response to virions and dense bodies of human cytomegalovirus determined by enzyme immunofluorescence assay.

An enzyme immunoassay utilizing a fluorogenic substrate (EIFA) was employed to compare humoral antibody responses to purified virion and dense body antigens in human cytomegalovirus (CMV) infections. Results of these antibody assays were also compared to those obtained by plaque reduction neutralization and complement fixation (CF). In adults with CMV infections diagnosed by a significant rise in CF antibody titer, there was also a vigorous antibody response to the virion and dense body antigens. There was better correlation between antibody levels to the virion and dense body antigens than between antibody levels detected by other combinations of tests. Antibody responses to the virion and dense body antigens were similar, but not identical, suggesting that although the two structures share major antigens, they may also possess unique antigens to which the host can mount independent antibody responses. All individuals with CMV neutralizing antibody also had virion and dense body antibodies, but there was poor correlation between neutralization titers and those detected by EIFA, perhaps reflecting greater strain specificity of the neutralization test. Some individuals with serological diagnoses of Mycoplasma pneumoniae or influenza A infection showed fourfold or greater antibody titer rises to CMV in two or more assays, suggesting reactivation of latent CMV in these infections.

Antibodies, Viral↗

Specific identification of human cytomegalovirus isolates by anti-complement immunofluorescence with immune hamster sera.

An anti-complement immunofluorescence test utilizing cytomegalovirus immune hamster sera specifically identified cytomegalovirus isolates showing an early, typical cytopathic effect. Inclusion of a control conjugate against adenovirus group antigen permitted correct identification, as adenoviruses, of a few isolates initially suspected of being cytomegalovirus on the basis of cytopathic effect.

Animals↗

Antigenic comparisons of two new rotaviruses from rhesus monkeys.

Two rotavirus strains isolated in cell culture from infant rhesus monkeys with diarrhea were closely related to SA 11 virus and to each other by plaque reduction neutralization tests. However, results of immune electron microscopy suggested possible antigenic differences between the two rhesus rotavirus strains.

Animals↗